Based on the importance of virus-specific CD8+ T cells in protective immunity, most of the current or recently completed HIV vaccine trials strive to elicit strong HIV-specific CTL activity. Yet, data from telomere studies on CD8+ T cells in HIV-infected persons suggest that the replicative limit of normal somatic cells may ultimately impede the continued protective function of these cells. This novel facet of memory CD8+ T cell biology -fundamental to vaccine efficacy, HIV pathogenesis, and immune reconstitution -is the theme of the current proposal. The major hallmarks of T cell replicative senescence identified in cell culture -shortened telomeres, absence of CD28, and inability to proliferate -are the precise features of a progressively increasing subset of CD8+ T cells found in HIV-infected individuals. This proposal will test the hypothesis that manipulation of the critical genes involved in signaling replicative senescence will stabilize telomere length, extend the replicative potential, and increase the function longevity of HIV-specific T cells without causing transformation or karyotypic abnormalities. The Specific Aims of the proposed research are: (1) To genetically modify HIV-specific CD8+ T cells in order to prevent telomere shortening and replicative senescence. Using retrovirally-mediated gene transfer, hTERT (the gene for the catalytic component of the human enzyme telomerase) and CD28 will be reintroduced into the T cells. The transduced cultures will be compared to vector alone and unmanipulated cultures for population doublings, telmerase activity, and telomere length. (2) To verify that the anti-viral protective functions of the genetically modified T cells are retained and that the cells show no evidence of transformation. Functional evaluation will include assays for antigen-specific cytotoxicity, viral suppressor factor production, and chemokine/cytokine production. Karyotypic analysis for chromosomal changes, cell cycle analysis for checkpoint integrity, and injection into SCID mice will be performed to verify that the manipulation of replicative senescence does not induce transformation. These studies provide a novel and innovative strategy for enhancing virus-specific long-term memory.